In the life sciences in general, and in biotechnology in particular there exists an extensive need for bioanalytical methods that enable the precise, fast and efficient analysis of chemical, biochemical end biological parameters in different sample formats, e.g. aqueous solutions, complex biochemical mixtures at the surface or in the interior of cells, or in specimens taken from plant or animal tissue.
The general task of such bioanalytical methods is the characterization of a biological or biochemical sample with regard to its chemical composition with a very high spatial and temporal resolution. Ideally, these methods allow the real-time analyses of the chemical composition of small, addressable volume elements and their change over time. The scale of such volume elements should be in the range of the microstructure of biological processes, i.e. the size of a cell and below. The temporal resolution should be in the order of the biological processes, i.e. in the range of microseconds and less. The sensitivity should be down to the single molecule level, because biological processes often rely on or are triggered by a single molecule. Moreover, populations of biological molecules—such as proteins—are obviously quite heterogeneous as can be seen from single molecule analyses, and this intrinsic heterogeneity seems to be quite important for the regulation of biological processes.
The use of fluorescence is extraordinarily well suited for such bioanalytical methods for a number of reasons. First, fluorescence is induced. Hence, molecules of interest can selectively be excited, thereby enabling to discriminate between different molecules and to suppress background. Second, fluorescence is a property of the single molecule, i.e. a single molecule can be excited and correspondingly emits single photons that can be detected. Therefore, fluorescence can be measured with single-molecule level sensitivity. Third, fluorescence parameters are characteristics of the nature of the molecule that undergoes fluorescence. The wavelength of photons that are absorbed, the lifetime of the excited state, the wavelength of emitted photons and the polarization angle of emitted photons are all characteristics of the chemical structure of the fluorescent molecule. These and more parameters can all be exploited to detect and discriminate different biomolecules. Fourth, fluorescence is fast. The lifetime of the fluorescent state is typically in the order of pico- to nanoseconds. Therefore, kinetics in the order of micro- to milliseconds can be resolved. Fifth, fluorescence is non-destructive and non-invasive and does not require any fixation of the specimen. Therefore, fluorescence can easily be analyzed in living cells or in tissue under mild conditions.
A number of bioanalytical methods that base on the measurement of fluorescence is known. In order to have a high spatial resolution, fluorimetry was rather early combined with confocal setups. The application of fluorimetry in a confocal setup in biotechnology was extensively demonstrated by Elgen and Rigler (PNAS 1994, 91(13):5740). Elgen and Rigler described in particular fluorescence correlation spectroscopy, which records correlations among the fluctuating fluorescence emission signal, and which thereby allows the analysis of molecular diffusion and transport phenomena. The authors used this technique for the measurement of molecular sizes and of biomolecular interactions such as the binding of a ligand to its receptor. Furthermore, they coupled these analyses with devices for trapping single molecules in electric fields, in order to increase the sensitivity and to decrease the detection limit further. In these measurements, molecules were labeled with specific fluorophores and could be monitored at concentrations of 10−15 M and less.
Problems of this and other setups are among others the limited spatial resolution, comparable long recording times in order to obtain very high data quality, the requirement for overlaying two or more laser beams in order to excite more than one fluorophor, and the difficulties of aligning the pinhole to restrict the open volume element in the axial direction.
Multi-photon excitation has been used to decrease the size of the volume element further and to clearly restrict the volume element without requiring any pinhole. The principles of two-photon excitation in combination with high resolution microscopy were described by Denk et al. (Science, 246 (1990) 73–76). Multi-photon excitation has several substantial advantages especially for the use with biological samples and methods based on molecular fluctuations in a microscopic focus. Two-Photon excitation is restricted strictly to the focal area of a microscope objective due to its non-linear nature. The result of this is a very high contrast, a very high spatial resolution, and no need to use a spatial filtering element (“pinhole”) to confine the detection volume, which facilitates the alignment procedure. Further, problems such as photodestruction and photobleaching of the biomolecules or the marker molecules are reduced. Photodestruction and similar effects often lead to false or misinterpreted results.
A particular set-up for the use of two-photon excitation is described in WO 02/08732. Sample analysts marked by different fluorescent dyes having spectrally different fluorescence emissions are illuminated by one laser-wavelength by means of two-photon excitation. The fluorescence emissions are detected by two separate detection devices. The use of two-photon excitation ensures both, the identity of the excitation volumes for both dyes and avoids the use of a pinhole for the confinement of the measuring volume in both detection paths. The setup is, however, limited to cross-correlation and/or confocal fluorescence coincidence analyses, and reveals no spatial information.
Analyses with higher precision, higher sample throughput, and in addition spatial information about each sample, can be achieved when parallelizing these single-focus confocal fluorimetric setups. Preferably this can be done using setups with multiple foci.
Improved setups for fluorimetric analyses have been described that detect fluorescence in a multitude of volume elements. The patent specification U.S. Pat. No. 6,815,262 describes an apparatus and a method for carrying out laser-induced two-photon fluorescence correlation spectroscopy in parallel in a plurality of probe volumes. In this setup, a laser beam is consecutively focused via microscope objectives through a series of sample volumes, i.e. the light passing one sample volume is refocused into the next sample. Fluorescence is detected by a parallel arrangement of optical devices comprising lenses, filters and a photomultipiler for each sample. The setup is complex since it requires high quality optics for each focus. Furthermore, the excitation intensity decreases from sample to sample due to optical losses in the samples and optics alongside the serial arrangement. This leads to limitations when comparing resulting fluorescence signals from different samples. Furthermore, the setup generates foci in different samples. Therefore, the setup reveals no spatial information from the sample.
WO 01/40789 describes a setup for measuring fluorescence in a confocal setup in a multitude of focal points created in each sample. The technical set-up uses a multitude of optical fibers, a beam splitting device and focussing optics. The laser beam or any other light used for excitation is guided through a multitude of optical fibers. The output of fibers is directed via a dichroic mirror to a focusing optics which in turn is focusing the excitation light into a multitude of secondary fibers. The output of the secondary set of fibers is focused by another focusing optics into the sample creating a multitude of foci. The resulting fluorescence from molecules in the foci is then collected by the same optics into the secondary fibers. Then, after passing the dichroic mirror, the fluorescence is focused into a third multitude of fibers which finally directs the fluorescence light onto at least one detector device. To achieve diffraction limited high quality focal spots in the sample and confined detection volumes, which are essential for measurements based on molecular fluctuations, all fibers in the optical excitation path have to be monomode fibers. Besides the fact that the alignment procedure is very time consuming, the coupling of light into monomode fibers is subject to severe loss of intensity. This accounts for the excitation light as well as for the detected fluorescence, which lead to a significant reduction in the number of detected emission photons per excited molecule. In addition, this makes the setup incompatible with multi-photon excitation since the fibers cause a significant pulse-broadening of fs laser pulses, which are necessary for two-photon excitation. Using one-photon excitation in a multi-focal setup, however, leads to severe crosstalk between the foci due to excitation beyond the focal area, which further reduces the quality of the signal.
In summary, all the conventional setups described so far are limited in terms of their spatial and temporal resolution, their signal quality, their recording speed and therefore their sample throughput their robustness toward complex and changing sample matrices, and their technical stability and requirements for readjustments and alignments.